Energy transfer properties of Rhodobacter sphaeroides chromatophores during adaptation to low light intensity.

Time-resolved fluorescence spectroscopy was used to explore the pathway and kinetics of energy transfer in photosynthetic membrane vesicles (chromatophores) isolated from Rhodobacter (Rba.) sphaeroides cells harvested 2, 4, 6 or 24 hours after a transition from growth in high to low level illumination. As previously observed, this light intensity transition initiates the remodeling of the photosynthetic apparatus and an increase in the number of light harvesting 2 (LH2) complexes relative to light harvesting 1 (LH1) and reaction center (RC) complexes. It has generally been thought that the increase in LH2 complexes served the purpose of increasing the overall energy transmission to the RC. However, fluorescence lifetime measurements and analysis in terms of energy transfer within LH2 and between LH2 and LH1 indicate that, during the remodeling time period measured, only a portion of the additional LH2 generated are well connected to LH1 and the reaction center. The majority of the additional LH2 fluorescence decays with a lifetime comparable to that of free, unconnected LH2 complexes. The presence of large LH2-only domains has been observed by atomic force microscopy in Rba. sphaeroides chromatophores (Bahatyrova et al., Nature, 2004, 430, 1058), providing structural support for the existence of pools of partially connected LH2 complexes. These LH2-only domains represent the light-responsive antenna complement formed after a switch in growth conditions from high to low illumination, while the remaining LH2 complexes occupy membrane regions containing mixtures of LH2 and LH1-RC core complexes. The current study utilized a multi-parameter approach to explore the fluorescence spectroscopic properties related to the remodeling process, shedding light on the structure-function relationship of the photosynthetic assembles. Possible reasons for the accumulation of these largely disconnected LH2-only pools are discussed.

Highly heterogenous humoral immune response in Lyme disease patients revealed by broad machine learning-assisted antibody binding profiling with random peptide arrays.

Introduction: Lyme disease (LD), a rapidly growing public health problem in the US, represents a formidable challenge due to the lack of detailed understanding about how the human immune system responds to its pathogen, the Borrelia burgdorferi bacterium. Despite significant advances in gaining deeper insight into mechanisms the pathogen uses to evade immune response, substantial gaps remain. As a result, molecular tools for the disease diagnosis are lacking with the currently available tests showing poor performance. High interpersonal variability in immune response combined with the ability of the pathogen to use a number of immune evasive tactics have been implicated as underlying factors for the limited test performance. Methods: This study was designed to perform a broad profiling of the entire repertoire of circulating antibodies in human sera at the single-individual level using planar arrays of short linear peptides with random sequences. The peptides sample sparsely, but uniformly the entire combinatorial sequence space of the same length peptides for profiling the humoral immune response to a B.burg. infection and compare them with other diseases with etiology similar to LD and healthy controls. Results: The study revealed substantial variability in antibody binding profiles between individual LD patients even to the same antigen (VlsE protein) and strong similarity between individuals diagnosed with Lyme disease and healthy controls from the areas endemic to LD suggesting a high prevalence of seropositivity in endemic healthy control. Discussion: This work demonstrates the utility of the approach as a valuable analytical tool for agnostic profiling of humoral immune response to a pathogen.

Nanosecond fluorescence from chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum.

Single-photon counting techniques were used to measure the fluorescence decay from Rhodopseudomonas sphaeroides and Rhodospirillum rubrum chromatophores after excitation with a 25-ps, 600-nm laser pulse. Electron transfer was blocked beyond the initial radical-pair state (PF) by chemical reduction of the quinone that serves as the next electron acceptor. Under these conditions, the fluorescence decays with multiphasic kinetics and at least three exponential decay components are required to describe the delayed fluorescence. Weak magnetic fields cause a small increase in the decay time of the longest component. The components of the delayed fluorescence are similar to those found previously with isolated reaction centers. We interpret the multi-exponential decay in terms of two small (0.01-0.02 eV) relaxations in the free energy of PF, as suggested previously for reaction centers. From the initial amplitudes of the delayed fluorescence, it is possible to calculate the standard free-energy difference between the earliest resolved form of PF and the excited singlet state of the antenna complexes in R. rubrum strains S1 and G9. The free-energy gap is found to be about 0.10 eV. It also is possible to calculate the standard free-energy difference between PF and the excited singlet state of the reaction center bacteriochlorophyll dimer (P). Values of 0.17 to 0.19 eV were found in both R. rubrum strains and also in Rps. sphaeroides strain 2.4.1. This free-energy gap agrees well with the standard free-energy difference between PF and P determined previously for reaction centers isolated from Rps. sphaeroides strain R26. The temperature dependence of the delayed fluorescence amplitudes between 180 K and 295 K is qualitatively different in isolated reaction centers and chromatophores. However, the temperature dependence of the calculated standard free-energy difference between P* and PF is similar in reaction centers and chromatophores of Rps. sphaeroides. The different temperature dependence of the fluorescence amplitudes in reaction centers and chromatophores arises because the free-energy difference between P* and the excited antenna is dominated by the entropy change associated with delocalization of the excitation in the antenna. We conclude that the state PF is similar in isolated reaction centers and in the intact photosynthetic membrane. Chromatophores from Rps. sphaeroides strain R-26 exhibit an anomalous fluorescence component that could reflect heterogeneity in their antenna.

Nanosecond fluorescence from isolated photosynthetic reaction centers of Rhodopseudomonas sphaeroides.

The time-course of fluorescence from reaction centers isolated from Rhodopseudomonas sphaeroides was measured using single-photon counting techniques. When electron transfer is blocked by the reduction of the electron-accepting quinones, reaction centers exhibit a relatively long-lived (delayed) fluorescence due to back reactions that regenerate the excited state (P*) from the transient radical-pair state, PF. The delayed fluorescence can be resolved into three components, with lifetimes of 0.7, 3.2 and 11 ns at 295 K. The slowest component decays with the same time-constant as the absorbance changes due to PF, and it depends on both temperature and magnetic fields in the same way that the absorbance changes do. The time-constants for the two faster components of delayed fluorescence are essentially independent of temperature and magnetic fields. The fluorescence also includes a very fast (prompt) component that is similar in amplitude to that obtained from unreduced reaction centers. The prompt fluorescence presumably is emitted mainly during the period before the initial charge-transfer reaction creates PF from P*. From the amplitudes of the prompt and delayed fluorescence, we calculate an initial standard free-energy difference between P* and PF of about 0.16 eV at 295 K, and 0.05 eV at 80 K, depending somewhat on the properties of the solvent. The multiphasic decay of the delayed fluorescence is interpreted in terms of relaxations in the free energy of PF with time, totalling about 0.05 eV at 295 K, possibly resulting from nuclear movements in the electron-carriers or the protein.

Radical-pair energetics and decay mechanisms in reaction centers containing anthraquinones, naphthoquinones or benzoquinones in place of ubiquinone.

In reaction centers from Rhodobacter sphaeroides (formerly called Rhodopseudomonas sphaeroides), light causes an electron-transfer reaction that forms the radical pair state (P+I-, or PF) from the initial excited singlet state (P) of a bacteriochlorophyll dimer (P). Subsequent electron transfer to a quinone (Q) produces the state P+Q-. Back electron transfer can regenerate P from P+Q-, giving rise to 'delayed' fluorescence that decays with approximately the same lifetime as P+Q-. The free-energy difference between P+Q- and P can be determined from the initial amplitude of the delayed fluorescence. In the present work, we extracted the native quinone (ubiquinone) from Rps. sphaeroides reaction centers, and replaced it by various anthraquinones, naphthoquinones, and benzoquinones. We found a rough correlation between the halfwave reduction potential (E1/2) of the quinone used for reconstitution (as measured polarographically in dimethylformamide) and the apparent free energy of the state P+Q- relatively to P. As the E1/2 of the quinone becomes more negative, the standard free-energy gap between P+Q- and P decreases. However, the correlation is quantitatively weak. Apparently, the effective midpoint potentials (Em) of the quinones in situ depend subtly on interactions with the protein environment in the reaction center. Using the value of the Em for ubiquinone determined in native reaction centers as a reference, and the standard free energies determined for P+Q- in reaction centers reconstituted with other quinones, the effective Em values of 12 different quinones in situ are estimated. In native reaction centers, or in reaction centers reconstituted with quinones that give a standard free-energy gap of more than about 0.8 eV between P+Q- and P*, charge recombination from P+Q- to the ground state (PQ) occurs almost exclusively by a temperature-insensitive mechanism, presumably electron tunneling. When reaction centers are reconstituted with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 eV, part or all of the decay proceeds through a thermally accessible intermediate. There is a linear relationship between the log of the rate constant for the decay of P+Q- via the intermediate state and the standard free energy of P+Q-. The higher the free energy, the faster the decay. The kinetic and thermodynamic properties of the intermediate appear not to depend strongly on the quinone used for reconstitution, indicating that the intermediate is probably not simply an activated form of P+Q-.(ABSTRACT TRUNCATED AT 400 WORDS)

EPR, ENDOR, and special TRIPLE measurements of P(*+) in wild type and modified reaction centers from Rb. sphaeroides.

The influence of the protein environment on the primary electron donor, P, a bacteriochlorophyll a dimer, of reaction centers from Rhodobacter sphaeroides, has been investigated using electron paramagnetic resonance and electron nuclear double resonance spectroscopy. These techniques were used to probe the effects on P that are due to alteration of three amino acid residues, His L168, Asn L170, and Asn M199. The introduction of Glu at L168, Asp at L170, or Asp at M199 changes the oxidation/reduction midpoint potential of P in a pH-dependent manner (Williams et al. (2001) Biochemistry 40, 15403-15407). For the double mutant His L168 to Glu and Asn at L170 to Asp, excitation results in electron transfer along the A-side branch of cofactors at pH 7.2, but at pH 9.5, a long-lived state involving B-side cofactors is produced (Haffa et al. (2004) J Phys Chem B 108, 4-7). Using electron paramagnetic resonance spectroscopy, the mutants with alterations of each of the three individual residues and a double mutant, with changes at L168 and L170, were found to have increased linewidths of 10.1-11.0 G compared to the linewidth of 9.6 G for wild type. The Special TRIPLE spectra were pH dependent, and at pH 8, the introduction of aspartate at L170 increased the spin density ratio, rho (L)/rho (M), to 6.1 while an aspartate at the symmetry related position, M199, decreased the ratio to 0.7 compared to the value of 2.1 for wild type. These results indicate that the energy of the two halves of P changes by about 100 meV due to the mutations and are consistent with the interpretation that electrostatic interactions involving these amino acid residues contribute to the switch in pathway of electron transfer.

A fluorescence resonance energy transfer-based probe to monitor nucleosome structure.

Nucleosomes are the basic units of eukaryotic chromatin structure. By restricting factor access to regulatory DNA sequences, nucleosomes significantly impact genomic processes such as transcription, and various mechanisms to alter nucleosome structure to relieve this repression have evolved. Both nucleosomes and processes that alter them are inherently dynamic in nature. Thus, studies of dynamics will be necessary to truly understand these relief mechanisms. We describe here the characteristics of a novel fluorescence resonance energy transfer-based reporter that can clearly signal the formation of a canonical nucleosome structure and follow conformational and compositional changes in that structure, both at the ensemble-average (bulk) and at the single molecule level. Labeled nucleosomes behave conformationally and thermodynamically like typical nucleosomes; thus they are relevant reporters of nucleosome behavior. Nucleosomes and free DNA are readily distinguishable at the single-molecule level. Thus, these labeled nucleosomes are well suited to studies of dynamic changes in nucleosome structure including single-molecule dynamics.

Thermodynamics of specific and nonspecific DNA binding by two DNA-binding domains conjugated to fluorescent probes.

The complexes designed in this work combine the sequence-specific binding properties of helix-turn-helix DNA-binding motifs with intercalating cyanine dyes. Thermodynamics of the Hin recombinase and Tc3 transposase DNA-binding domains with and without the conjugated dyes were studied by fluorescence techniques to determine the contributions to specific and nonspecific binding in terms of the polyelectrolyte and hydrophobic effects. The roles of the electrostatic interactions in binding to the cognate and noncognate sequences indicate that nonspecific binding is more sensitive to changes in salt concentration, whereas the change in the heat capacity shows a greater sensitivity to temperature for the sequence-specific complexes in each case. The conjugated dyes affect the Hin DNA-binding domain by acting to anchor a short stretch of amino acids at the N-terminal end into the minor groove. In contrast, the N-terminal end of the Tc3 DNA-binding domain is bound in a well-ordered fashion to the DNA even in the absence of the conjugated dye. The conjugated dye and the DNA-binding domain portions of each conjugate bind noncooperatively to the DNA. The characteristic thermodynamic parameters of specific and nonspecific DNA binding by each of the DNA-binding domains and their respective conjugates are presented.

Fluorescent and photochemical properties of a single zinc finger conjugated to a fluorescent DNA-binding probe.

A single zinc finger derived from the DNA-binding domain of the glucocorticoid receptor (GR) has been tethered to the intercalating fluorophore thiazole orange, and the DNA recognition characteristics of the conjugate have been examined. DNA sequence specificity for the peptide-dye conjugate, determined by steady-state fluorescence measurements and photoactivated DNA cleavage experiments, reproduce the binding features of response element recognition found in the native GR. The thiazole orange is able to intercalate and fluoresce when the conjugate binds, at concentrations where little fluorescence is observed from either the conjugate alone or the conjugate mixed with DNA lacking the zinc finger target sequence. The conjugate preferentially targets a 5'-TGTTCT-3' sequence (the native glucocorticoid receptor element) with a dissociation constant of about 25 nM. Lower binding affinities (up to 10-fold) are observed for single site variants of this sequence, and much lower affinity (40-50-fold) is observed for binding to the estrogen response element (which differs from the glucocorticoid receptor element at two positions) as well as to nonspecific DNA. Footprinting reactions show a 4-6 base pair region that is protected by the zinc finger moiety. Photocleavage assays reveal a several base pair region flanking the recognition sequence where the tethered thiazole orange moiety is able to intercalate and subsequently cleave DNA upon visible light exposure. Thiazole orange is also shown to oxidize the 5'-G of remote GG sequences, depending on the details of the intervening DNA sequence. Small synthetic protein-dye conjugates such as this one are potentially useful for a variety of purposes including sequence-specific probes that work under physiological conditions (without melting and hybridization of DNA), sequence-specific photocleavage agents, and self-assembling components in electron and energy transfer systems that utilize DNA as a scaffold and/or photochemical medium.

Sequence homologies among E. coli ribosomal proteins: evidence for evolutionarily related groupings and internal duplications.

The complete or partial sequences of 47 E. coli ribosomal proteins described in the literature have been examined by computerized search and matching programs. In contrast to results previously reported by other investigators, sequence homologies were uncovered among some of these ribosomal proteins that are well beyond statistical expectations. Moreover, alignments of the most strongly homologous sequences suggested the existence of a network of family groupings. Several of these proteins also exhibit internal homologies, indicating that they have been elongated by a series of tandem duplications.

The identification and localization of 33 pea chloroplast transcription initiation sites.

We have used a novel approach to produce a comprehensive transcription initiation map of the pea chloroplast genome. Sites were mapped by measuring the ability of DNA probes to protect 5' ends of transcripts that have been capped in vitro. Using this approach, at least 33 probes appear to contain one or more transcription start sites. A more precise location of some of these sites was obtained by hybrid selecting certain of these RNAs and determining their size both before and after RNase treatment. We have found at least one initiation site in front of every chloroplast gene cluster for which appropriate clones were available. In addition, we have found initiation sites within gene clusters previously shown to be co-transcribed. In one such case, we were able to locate a transcription start site for psbC within the coding sequence of psbD.

Picosecond kinetics of the initial photochemical electron-transfer reaction in bacterial photosynthetic reaction centers.

The absorption changes that occur in reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides during the initial photochemical electron-transfer reaction have been examined. Measurements were made between 740 and 1300 nm at 295 and 80 K by using a pulse-probe technique with 610-nm, 0.8-ps flashes. An excited singlet state of the bacteriochlorophyll dimer P* was found to give rise to stimulated emission with a spectrum similar to that determined previously for fluorescence from reaction centers. The stimulated emission was used to follow the decay of P*; its lifetime was 4.1 +/- 0.2 ps at 295 K and 2.2 +/- 0.1 ps at 80 K. Within the experimental uncertainty, the absorption changes associated with the formation of a bacteriopheophytin anion, Bph-, develop in concert with the decay of P* at both temperatures, as does the absorption increase near 1250 nm due to the formation of the cation of P, P+. No evidence was found for the formation of a bacteriochlorophyll anion, Bchl-, prior to the formation of Bph-. This is surprising, because in the crystal structure of the Rhodopseudomonas viridis reaction center [Deisenhofer, J., Epp, O., Miki, K., Huber, R., & Michel, H. (1984) J. Mol. Biol. 180, 385-398] a Bchl is located approximately in between P and the Bph. It is possible that Bchl- (or Bchl+) is formed but, due to kinetic or thermodynamic constraints, is never present at a sufficient concentration for us to observe. Alternatively, a virtual charge-transfer state, such as P+Bchl-Bph or PBchl+Bph-, could serve to lower the energy barrier for direct electron transfer between P* and the Bph.

Photosynthetic reaction center mutagenesis via chimeric rescue of a non-functional Rhodobacter capsulatus puf operon with sequences from Rhodobacter sphaeroides.

Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3' of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3' end of pufM and sequences past the end of pufM.

Femtosecond pump-probe analysis of energy and electron transfer in photosynthetic membranes of Rhodobacter capsulatus.

Low-intensity, 295 K, femtosecond pump-probe transient absorption measurements are described that have been performed to investigate energy and electron transfer in photosynthetic membranes from a Rhodobacter capsulatus strain lacking functional light harvesting antenna complex II. Spectral and kinetic similarities between the absorption changes of isolated reaction centers and those of reaction centers in membranes upon 800-nm excitation suggest that the charge separation process in both cases is very similar. An ultrafast energy relaxation process observed near 872 nm when 800-nm excitation is used is interpreted as interexcitonic relaxation within the antenna, though other interpretations, such as vibrational relaxation, are possible. On the basis of global exponential fitting analysis of the time-dependent spectral changes using 800- and 880-nm excitation wavelengths to selectively excite the reaction center and the LHI antenna, respectively, it is found that excitation energy transfer and trapping in Rb. capsulatus is limited by the overall rate of energy transfer between the antenna and the reaction center. This conclusion is supported by the observation that excitation at 800 nm, but not 880 nm, results in absorbance changes indicative of charge separation with a lifetime (3.1 ps) very close to that reported for charge separation in isolated reaction centers (3.5 ps). Thus, most reaction centers that are directly excited undergo charge separation and not backward energy transfer to the LHI antenna complexes. Both a kinetic model analysis and a direct comparison between time-resolved spectra obtained using different excitation wavelengths resulted in an energy-detrapping efficiency of about 15 +/- 10%.

Characterization of a Rhodobacter capsulatus reaction center mutant that enhances the distinction between spectral forms of the initial electron donor.

A large scale mutation of the Rhodobacter capsulatus reaction center M-subunit gene, sym2-1, has been constructed in which amino acid residues M205-M210 have been changed to the corresponding L subunit amino acids. Two interconvertable spectral forms of the initial electron donor are observed in isolated reaction centers from this mutant. Which conformation dominates depends on ionic strength, the nature of the detergent used, and the temperature. Reaction centers from this mutant have a ground-state absorbance spectrum that is very similar to wild-type when measured immediately after purification in the presence of high salt. However, upon subsequent dialysis against a low ionic strength buffer or the addition of positively charged detergents, the near-infrared spectral band of P (the initial electron donor) in sym2-1 reaction centers is shifted by over 30 nm to the blue, from 852 to 820 nm. Systematically varying either the ionic strength or the amount of charged detergent reveals an isobestic point in the absorbance spectrum at 845 nm. The wild-type spectrum also shifts with ionic strength or detergent with an isobestic point at 860 nm. The large spectral separation between the two dominant conformational forms of the sym2-1 reaction center makes detailed measurements of each state possible. Both of the spectral forms of P bleach in the presence of light. Electrochemical measurements of the P/P+ midpoint potential of sym2-1 reaction centers show an increase of about 30 mV upon conversion from the long-wavelength form to the short-wavelength form of the mutant. The rate constant of initial electron transfer in both forms of the mutant reaction centers is essentially the same, suggesting that the spectral characteristics of P are not critical for charge separation. The short-wavelength form of P in this mutant also converts to the long-wavelength form as a function of temperature between room temperature and 130 K, again giving rise to an isobestic point, in this case at 838 nm for the mutant. A similar, though considerably less pronounced spectral change with temperature occurs in wild-type reaction centers, with an isobestic point at about 855 nm, close to that found by titrating with ionic strength or detergent. Fitting the temperature dependence of the sym2-1 reaction center spectrum to a thermodynamic model resulted in a value for the enthalpy of the conformational interconversion between the short- and long-wavelength forms of about -6 kJ/mol and an entropy of interconversion of about -35 J/(K mol). Similar values of enthapy and entropy changes can be used to model the temperature dependence in wild-type. Thus, much of the temperature dependence of the reaction center special pair near-infrared absorbance band can be described as an equilibrium shift between two spectrally distinct conformations of the reaction center.

Energy trapping and detrapping by wild type and mutant reaction centers of purple non-sulfur bacteria.

Time-correlated single photon counting was used to study energy trapping and detrapping kinetics at 295 K in Rhodobacter sphaeroides chromatophore membranes containing mutant reaction centers. The mutant reaction centers were expressed in a background strain of Rb. sphaeroides which contained only B880 antenna complexes and no B800-850 antenna complexes. The excited state decay times in the isolated reaction centers from these strains were previously shown to vary by roughly 15-fold, from 3.4 to 52 ps, due to differences in the charge separation rates in the different mutants (Allen and Williams (1995) J Bioenerg Biomembr 27: 275-283). In this study, measurements were also performed on wild type Rhodospirillum rubrum and Rb. sphaeroides B880 antenna-only mutant chromatophores for comparison. The emission kinetics in membranes containing mutant reaction centers was complex. The experimental data were analyzed in terms of a kinetic model that involved fast excitation migration between antenna complexes followed by reversible energy transfer to the reaction center and charge separation. Three emission time constants were identified by fitting the data to a sum of exponential decay components. They were assigned to trapping/quenching of antenna excitations by the reaction center, recombination of the P(+)H(-) charge-separated state of the reaction center reforming an emitting state, and emission from uncoupled antenna pigment-protein complexes. The first varied from 60 to 160 ps, depending on the reaction center mutation; the second was 200-300 ps, and the third was about 700 ps. The observed weak linear dependence of the trapping time on the primary charge separation time, together with the known sub-picosecond exciton migration time within the antenna, supports the concept that it is energy transfer from the antenna to the reaction center, rather than charge separation, that limits the overall energy trapping time in wild type chromatophores. The component due to charge recombination reforming the excited state is minor in wild type membranes, but increases substantially in mutants due to the decreasing free energy gap between the states P(*) and P(+)H(-).

Blue light drives B-side electron transfer in bacterial photosynthetic reaction centers.

The core of the photosynthetic reaction center from the purple non-sulfur bacterium Rhodobacter sphaeroides is a quasi-symmetric heterodimer, providing two potential pathways for transmembrane electron transfer. Past measurements have demonstrated that only one of the two pathways (the A-side) is used to any significant extent upon excitation with red or near-infrared light. Here, it is shown that excitation with blue light into the Soret band of the reaction center gives rise to electron transfer along the alternate or B-side pathway, resulting in a charge-separated state involving the anion of the B-side bacteriopheophytin. This electron transfer is much faster than normal A-side transfer, apparently occurring within a few hundred femtoseconds. At low temperatures, the B-side charge-separated state is stable for at least 1 ns, but at room temperature, the B-side bacteriopheophytin anion is short-lived, decaying within approximately 15 ps. One possible physiological role for B-side electron transfer is photoprotection, rapidly quenching higher excited states of the reaction center.

Comparative study of reaction centers from purple photosynthetic bacteria: Isolation and optical spectroscopy.

Reaction centers from two species of purple bacteria, Rhodospirillum rubrum and Rhodospirillum centenum, have been characterized and compared to reaction centers from Rhodobacter sphaeroides and Rhodobacter capsulatus. The reaction centers purified from these four species can be divided into two classes according to the spectral characteristics of the primary donor. Reaction centers from one class have a donor optical band at a longer wavelength, 865 nm compared to 850 nm, and an optical absorption band associated with the oxidized donor at 1250 nm that has a larger oscillator strength than reaction centers from the second class. Under normal buffering conditions, reaction centers isolated from Rb. sphaeroides and Rs. rubrum exhibit characteristics of the first class while those from Rb. capsulatus and Rs. centenum exhibit characteristics of the second class. However, the reaction centers can be converted between the two groups by the addition of charged detergents. Thus, the observed spectral differences are not due to intrinsic differences between reaction centers but represent changes in the electronic structure of the donor due to interactions with the detergents as has been confirmed by recent ENDOR measurements (Rautter J, Lendzian F, Lubitz W, Wang S and Allen JP (1994) Biochemistry 33: 12077-12084). The oxidation midpoint potential for the donor has values of 445 mV, 475 mV, 480 mV and 495 mV for Rs. rubrum, Rs. centenum, Rb. capsulatus, and Rb. sphaeroides, respectively. Despite this range of values for the midpoint potential, the decay rates of the stimulated emission are all fast with values of 4.1 ps, 4.5 ps. 5.5 ps and 6.1 ps for quinone-reduced RCs from Rs. rubrum, Rb. capsulatus, Rs. centenum, and Rb. sphaeroides, respectively. The general spectral features of the initial charge separated state are essentially the same for the four species, except for differences in the wavelengths of the absorption changes due to the different donor band positions. The pH dependence of the charge recombination rates from the primary and secondary quinones differ for reaction centers from the four species indicating different interactions between the quinones and ionizable residues. A different mechanism for charge recombination from the secondary quinone, that probably is direct recombination, is proposed for RCs from Rs. centenum.

Low-temperature femtosecond-resolution transient absorption spectroscopy of large-scale symmetry mutants of bacterial reaction centers.

Reaction centers isolated from three large-scale symmetry mutants sym0, sym2-1, and sym5-2 described in the previous article of this issue [Taguchi, A. K. W., Eastman, J. E., Gallo, D. M., Jr., Sheagley, E.. Xiao, W., & Woodbury, N. W. (1996) Biochemistry 35, 3175-3186] have been investigated by low-temperature ground state and ferntosecond-resolution transient absorption spectroscopy. All three of these large-scale symmetry mutants undergo electron transfer at 20 K. The mutants sym0 and sym5-2 have yields and dominant rates of charge separation comparable to wild type. However. the sym2-mutant shows a roughly 35%, quantum yield at this temperature, and the major kinetic component of the initial electron transfer is slower than wild type by nearly a factor of 100. The sym0 mutant showed substantial changes in the monomer bacteriochiorophyll ground state and transient spectra, and both sym0 sym2-1 showed changes in the bacteriopheophyll ground state and transient spectra. In particular, sym2-1 shows a small absorbance decrease in the region of the Qx band of the B side bacteriopheophytin which could be attributed to 10%-20% electron transfer along the B pathway.